Single-celled parasites from the phylum Apicomplexa place a global burden on human health. One of the best- studied Apicomplexans is Toxoplasma gondii, a ubiquitous pathogen that can cause life-threatening infections in the developing fetus and immune-deficient patients. Currently, there is no vaccine or cure for chronic infection and therapies for acute infection are limited and poorly tolerated. The related parasite Plasmodium causes human malaria. Though curative treatments exist for Plasmodium infections, parasites resistant to these drugs are on the rise. This study investigates a process that is conserved across Apicomplexans and therefore has the potential to uncover drug targets that apply to the entire phylum. Briefly, Apicomplexan parasites must invade various cell types in warm-blooded hosts and establish a protected replicative niche in order to survive. To do this, they inject effector proteins from specialized organelles called rhoptries directly into the host cell. To gain access to the host cytosol, the rhoptry proteins must cross three membrane barriers, an unusual and nontrivial topological problem that is rare in eukaryotes. Despite its crucial role in the biology and survival of these parasites, the trigger, machinery, and molecular mechanism for the injection of rhoptry proteins into the host cell are unknown. Electron micrographs reveal an opening between the host and parasite plasma membranes (both for Toxoplasma and Plasmodium), at exactly the time and location expected for rhoptry protein release. This suggests that a novel rhoptry protein injection complex (RPIC) spans the host and parasite plasma membranes and the rhoptry membrane. The proposed studies will probe for and validate factors that mediate rhoptry protein injection using the model organism Toxoplasma gondii. An unbiased proteomic screen that relies on novel applications of existing technologies will be performed in parallel with a rational bioinformatic screen of the Toxoplasma genome to identify putative RPIC proteins. RPIC candidates will be validated based on their location within parasites and on their function in rhoptry protein injection, using cutting-edge genetic and molecular tools. The results of this study will reveal RPIC proteins and for the first time make mechanistic investigations of invasion and rhoptry protein injection possible. This will open up a novel arena of strategies to control Toxoplasma, Plasmodium, and other medically important Apicomplexan parasites.